This invention relates to novel protease inhibitors, particularly inhibitors of cysteine and serine proteases, more particularly compounds which inhibit cysteine proteases, even more particularly compounds which inhibit cysteine proteases of the papain superfamily, yet more particularly compounds which inhibit cysteine proteases of the cathepsin family, most particularly compounds which inhibit cathepsin K. Such compounds are particularly useful for treating diseases in which cysteine proteases are implicated, especially diseases of excessive bone or cartilage loss, e.g., osteoporosis, periodontitis, and arthritis.
Cathepsin K is a member of the family of enzymes which are part of the papain superfamily of cysteine proteases. Cathepsins B, H, L, N and S have been described in the literature. Recently, cathepsin K polypeptide and the cDNA encoding such polypeptide were disclosed in U.S. Pat. No. 5,501,969 (called cathepsin O therein). Cathepsin K has been recently expressed, purified, and characterized. Bossard, M. J., et al., (1996) J. Biol. Chem. 271, 12517-12524; Drake, F. H., et al., (1996) J. Biol. Chem. 271, 12511-12516; Bromme, D., et al., (1996) J. Biol. Chem. 271, 2126-2132.
Cathepsin K has been variously denoted as cathepsin O, cathepsin X or cathepsin O2 in the literature. The designation cathepsin K is considered to be the more appropriate one (name assigned by Nomenclature Committee of the International Union of Biochemistry and Molecular Biology).
Cathepsins of the papain superfamily of cysteine proteases function in the normal physiological process of protein degradation in animals, including humans, e.g., in the degradation of connective tissue. However, elevated levels of these enzymes in the body can result in pathological conditions leading to disease. Thus, cathepsins have been implicated in various disease states, including but not limited to, infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei brucei, and Crithidia fusiculata; as well as in schistosomiasis malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like. See International Publication Number WO 94/04172, published on Mar. 3, 1994, and references cited therein. See also European Patent Application EP 0 603 873 A1, and references cited therein. Two bacterial cysteine proteases from P. gingivallis, called gingipains, have been implicated in the pathogenesis of gingivitis. Potempa, J., et al. (1994) Perspectives in Drug Discovery and Design, 2, 445-458.
Cathepsin K is believed to play a causative role in diseases of excessive bone or cartilage loss. Bone is composed of a protein matrix in which spindle- or plate-shaped crystals of hydroxyapatite are incorporated. Type I Collagen represents the major structural protein of bone comprising approximately 90% of the structural protein. The remaining 10% of matrix is composed of a number of non-collagenous proteins, including osteocalcin, proteoglycans, osteopontin, osteonectin, thrombospondin, fibronectin, and bone sialoprotein. Skeletal bone undergoes remodeling at discrete foci throughout life. These foci, or remodeling units, undergo a cycle consisting of a bone resorption phase followed by a phase of bone replacement.
Bone resorption is carried out by osteoclasts, which are multinuclear cells of hematopoietic lineage. The osteoclasts adhere to the bone surface and form a tight sealing zone, followed by extensive membrane ruffling on their apical (i.e., resorbing) surface. This creates an enclosed extracellular compartment on the bone surface that is acidified by proton pumps in the ruffled membrane, and into which the osteoclast secretes proteolytic enzymes. The low pH of the compartment dissolves hydroxyapatite crystals at the bone surface, while the proteolytic enzymes digest the protein matrix. In this way, a resorption lacuna, or pit, is formed. At the end of this phase of the cycle, osteoblasts lay down a new protein matrix that is subsequently mineralized. In several disease states, such as osteoporosis and Paget""s disease, the normal balance between bone resorption and formation is disrupted, and there is a net loss of bone at each cycle. Ultimately, this leads to weakening of the bone and may result in increased fracture risk with minimal trauma.
The abundant selective expression of cathepsin K in osteoclasts strongly suggests that this enzyme is essential for bone resorption. Thus, selective inhibition of cathepsin K may provide an effective treatment for diseases of excessive bone loss, including, but not limited to, osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget""s disease, hypercalcemia of malignancy, and metabolic bone disease. Cathepsin K levels have also been demonstrated to be elevated in chondroclasts of osteoarthritic synovium. Thus, selective inhibition of cathepsin K may also be useful for treating diseases of excessive cartilage or matrix degradation, including, but not limited to, osteoarthritis and rheumatoid arthritis. Metastatic neoplastic cells also typically express high levels of proteolytic enzymes that degrade the surrounding matrix. Thus, selective inhibition of cathepsin K may also be useful for treating certain neoplastic diseases.
It now has been discovered that a novel class of compounds are protease inhibitors, most particularly inhibitors of cathepsin K, and these compounds are useful for treating diseases in which inhibition of bone resorption is indicated, such as osteoporosis and periodontal disease.
An object of the present invention is to provide protease inhibitors, particularly such inhibitors of cysteine and serine proteases, more particularly such compounds which inhibit cysteine proteases, even more particularly such compounds which inhibit cysteine proteases of the papain superfamily, yet more particularly such compounds which inhibit cysteine proteases of the cathepsin family, most particularly such compounds which inhibit cathepsin K, and which are useful for treating diseases which may be therapeutically modified by altering the activity of such proteases.
Accordingly, in the first aspect, this invention provides a compound according to formula (I).
In another aspect, this invention provides a pharmaceutical composition comprising a compound according to formula (I) and a pharmaceutically acceptable carrier.
In yet another aspect, this invention provides a method of treating diseases in which the disease pathology may be therapeutically modified by inhibiting proteases, particularly cysteine and serine proteases, more particularly cysteine proteases, even more particularly cysteine proteases of the papain superfamily, yet more particularly cysteine proteases of the cathepsin family, most particularly cathepsin K.
In a particular aspect, the compounds of this invention are especially useful for treating diseases characterized by bone loss, such as osteoporosis and gingival diseases, such as gingivitis and periodontitis, or by excessive cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis.
The present invention provides compounds of formula (I): 
wherein:
Y is Ar or NR1R2;
R1 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), or Rxe2x80x3Rxe2x80x2NC(S);
R2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R3 is H, C2-6alkenyl, C2-6alkynyl, Het, Ar or C1-6alkyl optionally substituted by ORxe2x80x2, SRxe2x80x2, NRxe2x80x22, N(Rxe2x80x2)C(O)ORxe2x80x3, CO2Rxe2x80x2, CO2NRxe2x80x22, N(Cxe2x95x90NH)NH2, Het or Ar;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R5 is 
xe2x80x83Ar-C0-6alkyl, Het-C0-6alkyl, adamantyl-C(O)xe2x80x94, Ar-C(O)xe2x80x94, or Het-C(O)xe2x80x94;
R6 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), Rxe2x80x3Rxe2x80x2NC(S),or Rxe2x80x3OC(O)NRxe2x80x2CH(R*)C(O);
R7 is C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C0-6alkoxy, Het-C0-6alkoxy, or C1-6alkyl optionally substituted by ORxe2x80x2, SRxe2x80x2, NRxe2x80x22, N(Rxe2x80x2)C(O)ORxe2x80x3, CO2Rxe2x80x2, CO2NRxe2x80x22, N(Cxe2x95x90NH)NH2, Het or Ar;
R* is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
each Rxe2x80x2 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each Rxe2x80x3 independently is C1-6alkyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x2xe2x80x3 is H, C1-6alkyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Z is C(O) or CH2; and
n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof.
Preferably, the present invention provides compounds of formula (Ia): 
wherein:
R1 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), or Rxe2x80x3Rxe2x80x2NC(S);
R2 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R3 is H, C2-6alkenyl, C2-6alkynyl, Het, Ar or C1-6alkyl optionally substituted by ORxe2x80x2, SRxe2x80x2, NRxe2x80x22, N(Rxe2x80x2)C(O)ORxe2x80x3, CO2Rxe2x80x2, CO2NRxe2x80x22, N(Cxe2x95x90NH)NH2, Het or Ar;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R5 is 
xe2x80x83Ar-C0-6alkyl, Het-C0-6alkyl, adamantyl-C(O)xe2x80x94, Ar-C(O)xe2x80x94, or Het-C(O)xe2x80x94;
R6 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), Rxe2x80x3Rxe2x80x2NC(S),or Rxe2x80x3OC(O)NRxe2x80x2CH(R*)C(O);
R7 is C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C0-6alkoxy, Het-C0-6alkoxy, or C1-6alkyl optionally substituted by ORxe2x80x2, SRxe2x80x2, NRxe2x80x22, N(Rxe2x80x2)C(O)ORxe2x80x3, CO2Rxe2x80x2, CO2NRxe2x80x22, N(Cxe2x95x90NH)NH2, Het or Ar;
R* is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
each Rxe2x80x2 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each Rxe2x80x3 independently is C1-6alkyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x2xe2x80x3 is H, C1-6alkyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Z is C(O) or CH2; and
n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof.
The present invention includes all hydrates, solvates, complexes and prodrugs of the compounds of this invention. Prodrugs are any covalently bonded compounds which release the active parent drug according to formula (I) in vivo. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Inventive compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
The meaning of any substituent at any one occurrence in formula (I) or any subformula thereof is independent of its meaning, or any other substituent""s meaning, at any other occurrence, unless specified otherwise.
With respect to formula (I):
Suitably, R4 and Rxe2x80x2xe2x80x3 are each H and R3 is C1-6alkyl or C2-6alkenyl. Preferably, R3 is i-butyl.
Suitably, R5 is benzyl or 
xe2x80x83in which Rxe2x80x2is H, R7 is C1-6alkyl, preferably i-butyl, R6 is Rxe2x80x3OC(O), wherein Rxe2x80x3 is benzyl, and Z is CH2.
Suitably, Y is NR1R2, in which R2 is H and R1 is Rxe2x80x3C(O) or Rxe2x80x3OC(O), and Rxe2x80x3 in said R1 group is C1-6alkyl, Ar-C0-6alkyl or Het-C0-6alkyl, and, most preferably, Rxe2x80x3 is tert-butyl, 
Suitably, n is 1 or 2. Preferably, n is 1.
In one particular embodiment, the formula (Ia) compound of this invention is a compound of formula (Ib): 
In another embodiment, the formula (Ia) compound of this invention is a compound of formula (Ic): 
Specific representative compounds of this invention are:
3-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
1-benzyl-3-[[Nxcex1-(2quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
3-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
1-benzyl-3-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(benzyloxycarbonyl)-L-leuciny]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(tert-butoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
1-benzyl-(3R)-[[Nxcex1-(2-naphthyl)acetyl-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3R)-[[Nxcex1-(2-naphtylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3R)-[[Nxcex1-(3-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3R)-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3R)-[[Nxcex1-(3-isoquinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(2-naphthyl)acetyl-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(3-quinolinecarbonyl)-L-leucinyl]amino-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(3-isoquinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-4-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-piperidine;
1-benzyl-4-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-piperidine;
1-benzyl-4-[[Nxcex1-(benzyloxycarbonyl)-L-leucinyl]amino]-piperidine;
1-[3-(2-pyridyl)phenyl]-2-ethyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-[3-(2-pyridyl)phenyl]-2-ethyl-(3S)-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-[3-(2-pyridyl)phenyl]-2-ethyl-(3S)-[[Nxcex1-(3-isoquinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-[3-(2-pyridyl)phenyl]-2-ethyl-(3R)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-[3-(2-pyridyl)phenyl]-2-ethyl-(3R)-[[Nxcex1-(3-isoquinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-[3-(2-pyridyl)phenyl]-2-ethyl-(3R)-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(1-adamantanecarbonyl)-(3R)-[[Nxcex1-(4-pyridylmethoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(1-adamantanecarbonyl)-(3S)-[[Nxcex1-(4-pyridylmethoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
(3R)-[[Nxcex1-(benzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(3,4-dimethoxybenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(benzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(benzothiazole-6-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(indole-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(4-fluorobenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(4-methoxybenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(3,4-dichlorobenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(thiophene-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(4-biphenylcarbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[N-(5-methoxybenzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(5-chlorobenzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(7-methoxybenzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(3-chlorobenzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3R)-[[Nxcex1-(3-(2-pyridyl)benzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(benzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(3,4-dimethoxybenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(benzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(benzothiazole-6-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(indole-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(4-fluorobenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(4-methoxybenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(3,4-dichlorobenzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(thiophene-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(4-biphenylcarbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(5-methoxybenzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(5-chlorobenzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(7-methoxybenzofuran-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(3-chlorobenzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
(3S)-[[Nxcex1-(3-(2-pyridyl)benzoyl)-L-leucinyl]amino]-1-[(2S)-4-methyl-2-[[(benzyloxycarbonyl)amino]pentyl]-pyrrolidine;
1-(4-phenyl)benzyl-(3S)-[[Nxcex1-(tert-butoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenyl)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenyl)benzyl-(3S)-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenyl)benzyl-(3S)-[[Nxcex1-(3,4-dimethoxybenzoyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenyl)benzyl-(3S)-[[Nxcex1-(benzofuran-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenyl)benzyl-(3S)-[[Nxcex1-(benzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenyl)benzyl-(3S)-[[Nxcex1-(benzyloxycarbonyl)-L-leucinyl]amino]-pyrrolidine,
1-(2-phenyl)ethyl-(3S)-[[Nxcex1-(tert-butoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-phenyl)ethyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-phenyl)ethyl-(3S)-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-phenyl)ethyl-(3S)-[[Nxcex1-(benzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-phenyl)ethyl-(3S)-[[Nxcex1-(benzofuran-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-phenyl)ethyl-(3S)-[[Nxcex1-(3-chlorobenzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenoxy)benzyl-(3S)-[[Nxcex1-(tert-butoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenoxy)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenoxy)benzyl-(3S)-[[Nxcex1-(2-quinolinecarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenoxy)benzyl-(3S)-[[Nxcex1-(3,4-dimethoxybenzoyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-phenoxy)benzyl-(3S)-[[Nxcex1-(benzofuran-2-carbonyl)-L-leucinyl]amino]-pyrrolidine
1-(4-phenoxy)benzyl-(3S)-[[Nxcex1-(benzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-fluoro)benzyl-(3S)-[[Nxcex1-(tert-butoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-fluoro)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-fluoro)benzyl-(3S)-[[Nxcex1-(benzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-cyano)benzyl-(3S)-[[Nxcex1-(tert-butoxycarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-cyano)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(benzo[b]thiophene-2-carbonyl)-L-leucinyl]amino]-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(3,4-dimethoxybenzoyl)-L-leucinyl]amino-pyrrolidine;
1-benzyl-(3S)-[[Nxcex1-(3-(2-dimethylaminoethoxy)-4-methoxybenzoyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-nitro)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-(N,N-dimethylamino)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-methoxy)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-pyridyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1]-(4-carboxymethyl)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3,4-methylenedioxy)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-naphthyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-indolyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-quinolinyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-quinolinyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(1-naphthyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-quinolinyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-pyrrolyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-pyridyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-pyridyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-nitro)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-acetamido)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-cyano)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-fluoro)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-phenoxy)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-chloro)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-trifluoromethyl)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-trifluoromethyl)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-(3-(N,N-dimethylamino)propoxy)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(4-(isopropyl)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-benzofuranyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-(3-methylbenzo[b]thiophenyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-furanyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3-furanyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-thiophenyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(2-nitro)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
-(3-thiophenyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
1-(3,4-dimethoxy)benzyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine; and
1-(5-nitro-3-furanyl)methyl-(3S)-[[Nxcex1-(2-naphthylcarbonyl)-L-leucinyl]amino]-pyrrolidine;
or a pharmaceutically acceptable salt thereof.
In yet another aspect, this invention provides novel intermediates useful in the preparation of formula (I) compounds represented by the formula (II): 
wherein:
R3 is H, C2-6alkenyl, C2-6alkynyl, Het, Ar or C1-6alkyl optionally substituted by ORxe2x80x2, SRxe2x80x2, NRxe2x80x22, N(Rxe2x80x2)C(O)ORxe2x80x3, CO2Rxe2x80x2, CO2NRxe2x80x22, N(Cxe2x95x90NH)NH2, Het or Ar;
R4 is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
R5 is 
xe2x80x83Ar-C0-6alkyl, Het-C0-6alkyl, adamantyl-C(O)xe2x80x94, Ar-C(O)xe2x80x94, or Het-C(O)xe2x80x94;
R6 is Rxe2x80x3, Rxe2x80x3C(O), Rxe2x80x3C(S), Rxe2x80x3SO2, Rxe2x80x3OC(O), Rxe2x80x3Rxe2x80x2NC(O), Rxe2x80x3Rxe2x80x2NC(S),or Rxe2x80x3OC(O)NRxe2x80x2CH(R*)C(O);
R7 is C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, Het-C0-6alkyl, Ar-C0-6alkoxy, Het-C0-6alkoxy, or C1-6alkyl optionally substituted by ORxe2x80x2, SRxe2x80x2, NRxe2x80x22, N(Rxe2x80x2)C(O)ORxe2x80x3, CO2Rxe2x80x2, CO2NRxe2x80x22, N(Cxe2x95x90NH)NH2, Het or Ar;
R* is H, C1-6alkyl, C2-6alkenyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, Het-C0-6alkyl;
each Rxe2x80x2 independently is H, C1-6alkyl, C2-6alkenyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
each Rxe2x80x3 independently is C1-6alkyl, C3-6cycloalkyl-C0-6-alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Rxe2x80x2xe2x80x3 is H, C1-6alkyl, C3-6cycloalkyl-C0-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl;
Z is C(O) or CH2; and
n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof.
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of the present invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984). The term xe2x80x9camino acidxe2x80x9d as used herein refers to the D- or L-isomers of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
xe2x80x9cC1-6alkylxe2x80x9d as applied herein is meant to include substituted and unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. Any C1-6alkyl group may be optionally substituted independently by one or two halogens, SRxe2x80x2, ORxe2x80x2, N(Rxe2x80x2)2, C(O)N(Rxe2x80x2)2, carbamyl or C1-4alkyl, where Rxe2x80x2 is H or C1-6alkyl. C0alkyl means that no alkyl group is present in the moiety. Thus, Ar-C0alkyl is equivalent to Ar.
xe2x80x9cC3-6cycloalkylxe2x80x9d as applied herein is meant to include substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane, and cyclohexane.
xe2x80x9cC2-6 alkenylxe2x80x9d as applied herein means an alkyl group of 2 to 6 carbons wherein a carbon-carbon single bond is replaced by a carbon-carbon double bond. C2-6alkenyl includes ethylene, 1-propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included.
xe2x80x9cC2-6alkynylxe2x80x9d means an alkyl group of 2 to 6 carbons wherein one carbon-carbon single bond is replaced by a carbon-carbon triple bond. C2-6 alkynyl includes acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne.
xe2x80x9cHalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d means F, Cl, Br, and I.
xe2x80x9cArxe2x80x9d or xe2x80x9carylxe2x80x9d means unsubstituted phenyl or naphthyl; or phenyl or naphthyl substituted by one or more of Ph-C0-6alkyl, Het-C0-6alkyl, C1-6alkoxy, Ph-C0-6alkoxy, Het-C0-6alkoxy, OH, (CH2)1-6NRxe2x80x2Rxe2x80x2, O(CH2)1-6NRxe2x80x2Rxe2x80x2; wherein each Rxe2x80x2 independently is H, C1-6alkyl, Ar-C0-6alkyl, or Het-C0-6alkyl; or phenyl or naphthyl substituted by one to three moieties selected from C1-4alkyl, ORxe2x80x2, N(Rxe2x80x2)2, SRxe2x80x2, CF3, NO2, CN, CO2Rxe2x80x2, CON(Rxe2x80x2), F, Cl, Br and I, or substituted by a methylenedioxy group.
As used herein xe2x80x9cHetxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d or xe2x80x9cheteroarylxe2x80x9d represents a stable 5- to 7-membered monocyclic or a stable 7- to 10-membered bicyclic heterocyclic ring, which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure, and may optionally be substituted with one or two moieties selected from C1-4alkyl, ORxe2x80x2, N(Rxe2x80x2)2, SRxe2x80x2, CF3, NO2, CN, CO2Rxe2x80x2, CON(Rxe2x80x2), F, Cl, Br and I, where Rxe2x80x2 is as defined hereinbefore. Examples of such heterocycles include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, pyranyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzoxazolyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, oxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzisoxazolyl, pyrimidinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,5-napthyridinyl, 1,6-napthyridinyl, 1,7-napthyridinyl, 1,8-napthyridinyl, tetrazolyl, 1,2,3-triazolyl, and 1,2,4-triazolyl. xe2x80x9cHetxe2x80x9d also means any heterocyclic moiety encompassed by the above definition of Het which is aromatic in character, e.g., pyridinyl, quinolinyl, isoquinolinyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, furyl, thienyl, benzoxazolyl, oxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzisoxazolyl, pyrimidinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,5-napthyridinyl, 1,6-napthyridinyl, 1,7-napthyridinyl, 1,8-napthyridinyl, tetrazolyl, 1,2,3-triazolyl, and 1,2,4-triazolyl.
Certain radical groups are abbreviated herein. t-Bu refers to the tertiary butyl radical, Boc or BOC refers to the t-butyloxycarbonyl radical, Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz or CBZ refers to the benzyloxycarbonyl radical.
Certain reagents are abbreviated herein. DCC refers to dicyclohexylcarbodiimide, DMAP is 2,6-dimethylaminopyridine, EDC or EDCI refers to N-ethyl-Nxe2x80x2(dimethylaminopropyl)-carbodiimide. HOBT or HOBt refers to 1-hydroxybenzotriazole, DMF refers to dimethyl formamide, BOP refers to benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate, DMAP is dimethylaminopyridine, DIEA refers to di-isopropylethylamine, Lawesson""s reagent is 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide, NMM is N-methylmorpholine, TFA refers to trifluoroacetic acid, TFAA refers to trifluoroacetic anhydride, KHMDS refers to potassium hexamethyldisilazide, and THF refers to tetrahydrofuran. Jones reagent is a solution of chromium trioxide, water, and sulfuric acid well-known in the art.
Compounds of the formula (I) are generally prepared by reacting a compound of the formula (II): 
or a salt thereof,
wherein Rxe2x80x2xe2x80x3, R3, R4, R5 and n are as defined in formula (I), with any reactive functional groups protected, with:
(a) Rxe2x80x3C(O)Cl, in which Rxe2x80x3 is as defined in formula (I) of claim 1; or
(b) Rxe2x80x3C(O)OH, in which Rxe2x80x3 is as defined in formula (I) of claim 1, in the presence of EDC and HOBT; or
(c) Rxe2x80x3C(O)H, in which Rxe2x80x3 is as defined in formula (I) of claim 1, followed by reduction; or
(d) Rxe2x80x3OC(O)Cl, in which Rxe2x80x3is as defined in formula (I) of claim 1, in the presence of base; or
(e) Rxe2x80x3SO2Cl, in which Rxe2x80x3 is as defined in formula (I) of claim 1, in the presence of base;
and thereafter removing any protecting groups and optionally forming a pharmaceutically acceptable salt.
Compounds of the formula (I) are prepared by methods analogous to those described in Schemes 1 and 2. 
a) PhCHO, CH2Cl2, NaBH(OAc)3; b) HCl, EtOAc, CH3OH; c) N-BOC-leucine, EDC, HOBt, NMM, CH2Cl2; d) HCl, EtOAc, CH3OH; e) quinaldic acid, EDC, HOBt, NMM, CH2Cl2 
Compounds of the general formula (1) wherein n is 1, R5 is an alkyl group and R1 is an Rxe2x80x2C(O) can be prepared as outlined in Scheme 1. Reductive alkylation of the commercially available amine 1-Scheme-1 (this material available in racemic or enantiomerically pure form) with an aldehyde, such as benzaldehyde or CBZ-leucinal, follwed by treatment with a reducing agent, such as sodium triacetoxyborohydride, affords the tertiary amine 2-Scheme-1. Removal of the protecting group by treating 2-Scheme-1 with a strong acid, such as hydrogen chloride, in ethyl acetate or ether or dioxane and methanol affords 3-Scheme-1. 3-Scheme-1 may be coupled with an acid using EDC and HOBT in the presence of a base, such as N-methylmorpholine or triethylamine, in an aprotic solvent, such as dichloromethane, to yield 4-Scheme-1. The protecting group of 4-Scheme-1 may be removed with strong acid, such as hydrogen chloride, in ethyl acetate or ether or dioxane and methanol to afford 5-Scheme-1. Coupling of the amine salt 5-Scheme-1 may be effected with an acid in the presence of EDC, HOBt and a base, such as N-methylmorpholine, to yield 6-Scheme-1. The 5-Scheme-1 salt may also be converted to the sulphonamide derivative by treatment with a sulphonyl chloride in the presence of a base, such as triethylamine, in an aprotic solvent, such as dichioromethane. 
a) N-BOC-leucine, EDC, HOBT, NMM, CH2Cl2; b) HCl, EtOAc; c) 2-naphthoic acid, EDC, HOBT, CH2Cl2, NMM
Compounds of the general formula (I) wherein n is 2, R5 is a benzyl group and R1 is an Rxe2x80x2 C(O) can be prepared as outlined in Scheme 1. Acylation of the commercially available 4-amino-1-benzylpiperidine (1-Scheme-1) with N-BOC-leucine in the presence of EDC, HOBT and N-methylmorpholine in dichloromethane afforded 2-scheme-2. Removal of the protecting group with anhydrous hydrogen chloride in ethyl acetate or ether or dioxane and methanol gave 3-Scheme-2. Acylation of the amine salt 3-Scheme-2 with a carboxylic acid as described previously afforded 4-Scheme-2.
The starting materials used herein are commercially available amino acids or are prepared by routine methods well known to those of ordinary skill in the art and can be found in standard reference books, such as the COMPENDIUM OF ORGANIC SYNTHETIC METHODS, Vol. I-VI (published by Wiley-Interscience).
Coupling methods to form amide bonds herein are generally well known to the art. The methods of peptide synthesis generally set forth by Bodansky et al., THE PRACTICE OF PEPTIDE SYNTHESIS, Springer-Verlag, Berlin, 1984; E. Gross and J. Meienhofer, THE PEPTIDES, Vol. 1, 1-284 (1979); and J. M. Stewart and J. D. Young, SOLID PHASE PEPTIDE SYNTHESIS, 2d Ed., Pierce Chemical Co., Rockford, Ill., 1984. are generally illustrative of the technique and are incorporated herein by reference.
Synthetic methods to prepare the compounds of this invention frequently employ protective groups to mask a reactive functionality or minimize unwanted side reactions. Such protective groups are described generally in Green, T. W, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley and Sons, New York (1981). The term xe2x80x9camino protecting groupsxe2x80x9d generally refers to the Boc, acetyl, benzoyl, Fmoc and Cbz groups and derivatives thereof as known to the art. Methods for protection and deprotection, and replacement of an amino protecting group with another moiety are well known.
Acid addition salts of the compounds of formula (I) are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts. Halides, sulfate, phosphate, alkanoates (such as acetate and trifluoroacetate), benzoates, and sulfonates (such as mesylate) are examples of anions present in pharmaceutically acceptable salts.
This invention also provides a pharmaceutical composition which comprises a compound according to formula (I) and a pharmaceutically acceptable carrier, diluent or excipient. Accordingly, the compounds of formula (I) may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of formula (I) prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
The compounds of formula (I) are useful as protease inhibitors, particularly as inhibitors of cysteine and serine proteases, more particularly as inhibitors of cysteine proteases, even more particularly as inhibitors of cysteine proteases of the papain superfamily, yet more particularly as inhibitors of cysteine proteases of the cathepsin family, most particularly as inhibitors of cathepsin K. The present invention also provides useful compositions and formulations of said compounds, including pharmaceutical compositions and formulations of said compounds.
The present compounds are useful for treating diseases in which cysteine proteases are implicated, including infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei, and Crithidia fusiculata; as well as in schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy; and especially diseases in which cathepsin K is implicated, most particularly diseases of excessive bone or cartilage loss, including osteoporosis, gingival disease including gingivitis and periodontitis, arthritis, more specifically, osteoarthritis and rheumatoid arthritis, Paget""s disease; hypercalcemia of malignancy, and metabolic bone disease.
Metastatic neoplastic cells also typically express high levels of proteolytic enzymes that degrade the surrounding matrix, and certain tumors and metastatic neoplasias may be effectively treated with the compounds of this invention.
The present invention also provides methods of treatment of diseases caused by pathological levels of proteases, particularly cysteine and serine proteases, more particularly cysteine proteases, even more particularly as inhibitors of cysteine proteases of the papain superfamily, yet more particularly cysteine proteases of the cathepsin family, which methods comprise administering to an animal, particularly a mammal, most particularly a human in need thereof a compound of the present invention. The present invention especially provides methods of treatment of diseases caused by pathological levels of cathepsin K, which methods comprise administering to an animal, particularly a mammal, most particularly a human in need thereof an inhibitor of cathepsin K, including a compound of the present invention. The present invention particularly provides methods for treating diseases in which cysteine proteases are implicated, including infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei, and Crithidia fusiculata; as well as in schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and especially diseases in which cathepsin K is implicated, most particularly diseases of excessive bone or cartilage loss, including osteoporosis, gingival disease including gingivitis and periodontitis, arthritis, more specifically, osteoarthritis and rheumatoid arthritis, Paget""s disease, hypercalcemia of malignancy, and metabolic bone disease.
This invention further provides a method for treating osteoporosis or inhibiting bone loss which comprises internal administration to a patient of an effective amount of a compound of formula (I), alone or in combination with other inhibitors of bone resorption, such as bisphosphonates (i.e., allendronate), hormone replacement therapy, anti-estrogens, or calcitonin. In addition, treatment with a compound of this invention and an anabolic agent, such as bone morphogenic protein, iproflavone, may be used to prevent bone loss or to increase bone mass.
For acute therapy, parenteral administration of a compound of formula (I) is preferred. An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit cathepsin K. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
The compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.
No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.
The compounds of this invention may be tested in one of several biological assays to determine the concentration of compound which is required to have a given pharmacological effect.
Determination of Cathepsin K Proteolytic Catalytic Activity
All assays for cathepsin K were carried out with human recombinant enzyme. Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically Cbz-Phe-Arg-AMC, and were determined in 100 mM Na acetate at pH 5.5 containing 20 mM cysteine and 5 mM EDTA. Stock substrate solutions were prepared at concentrations of 10 or 20 mM in DMSO with 20 uM final substrate concentration in the assays. All assays contained 10% DMSO. Independent experiments found that this level of DMSO had no effect on enzyme activity or kinetic constants. All assays were conducted at ambient temperature. Product fluorescence (excitation at 360 nM; emission at 460 nM) was monitored with a Perceptive Biosystems Cytofluor II fluorescent plate reader. Product progress curves were generated over 20 to 30 minutes following formation of AMC product.
Inhibition Studies
Potential inhibitors were evaluated using the progress curve method. Assays were carried out in the presence of variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of inhibitor and substrate. Data analysis was conducted according to one of two procedures depending on the appearance of the progress curves in the presence of inhibitors. For those compounds whose progress curves were linear, apparent inhibition constants (Ki,app) were calculated according to equation 1 (Brandt et al., Biochemitsry, 1989, 28, 140):
v 32 VmA/[Ka(1+I/Ki, app)+A]xe2x80x83xe2x80x83(1)
where v is the velocity of the reaction with maximal velocity Vm, A is the concentration of substrate with Michaelis constant of Ka, and I is the concentration of inhibitor.
For those compounds whose progress curves showed downward curvature characteristic of time-dependent inhibition, the data from individual sets was analyzed to give kobs according to equation 2:
[AMC]=vsst+(v0xe2x88x92vss)[1xe2x88x92exp(xe2x88x92kobst)]/kobsxe2x80x83xe2x80x83(2)
where [AMC] is the concentration of product formed over time t, v0 is the initial reaction velocity and vss is the final steady state rate. Values for kobs were then analyzed as a linear function of inhibitor concentration to generate an apparent second order rate constant (kobs/inhibitor concentration or kobs/[I]) describing the time-dependent inhibition. A complete discussion of this kinetic treatment has been fully described (Morrison et al., Adv. Enzymol. Relat. Areas Mol. Biol., 1988, 61, 201).
One skilled in the art would consider any compound with a Ki of less than 50 micromolar to be a potential lead compound. Preferably, the compounds used in the method of the present invention have a Ki value of less than 1 micromolar. Most preferably, said compounds have a Ki value of less than 100 nanomolar. 4-(R,S)-Amino-N-[(8-quinolinesulfonyl)-S-leucine]-3-tetrahydrofuran-3-one, a compound of formula (I), has a Ki value that is greater than 10 micromolar.
Human Osteoclast Resorption Assay
Aliquots of osteoclastoma-derived cell suspensions were removed from liquid nitrogen storage, warmed rapidly at 37xc2x0 C. and washed xc3x971 in RPMI-1640 medium by centrifugation (1000 rpm, 5 min at 4xc2x0 C.). The medium was aspirated and replaced with murine anti-HLA-DR antibody, diluted 1:3 in RPMI-1640 medium, and incubated for 30 min on ice The cell suspension was mixed frequently.
The cells were washed xc3x972 with cold RPMI-1640 by centrifugation (1000 rpm, 5 min at 4xc2x0 C.) and then transferred to a sterile 15 mL centrifuge tube. The number of mononuclear cells were enumerated in an improved Neubauer counting chamber.
Sufficient magnetic beads (5/mononuclear cell), coated with goat anti-mouse IgG, were removed from their stock bottle and placed into 5 mL of fresh medium (this washes away the toxic azide preservative). The medium was removed by immobilizing the beads on a magnet and is replaced with fresh medium.
The beads were mixed with the cells and the suspension was incubated for 30 min on ice. The suspension was mixed frequently. The bead-coated cells were immobilized on a magnet and the remaining cells (osteoclast-rich fraction) were decanted into a sterile 50 mL centrifuge tube. Fresh medium was added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process was repeated xc3x9710. The bead-coated cells were discarded.
The osteoclasts were enumerated in a counting chamber, using a large-bore disposable plastic pasteur pipette to charge the chamber with the sample. The cells were pelleted by centrifugation and the density of osteoclasts adjusted to 1.5xc3x97104/mL in EMEM medium, supplemented with 10% fetal calf serum and 1.7 g/liter of sodium bicarbonate. 3 mL aliquots of the cell suspension (per treatment) were decanted into 15 mL centrifuge tubes. These cells were pelleted by centrifugation. To each tube 3 mL of the appropriate treatment was added (diluted to 50 uM in the EMEM medium). Also included were appropriate vehicle controls, a positive control (87MEM1 diluted to 100 ug/mL) and an isotype control (IgG2a diluted to 100 ug/mL). The tubes were incubate at 37xc2x0 C. for 30 min.
0.5 mL aliquots of the cells were seeded onto sterile dentine slices in a 48-well plate and incubated at 37xc2x0 C. for 2 h. Each treatment was screened in quadruplicate. The slices were washed in six changes of warm PBS (10 mL/well in a 6-well plate) and then placed into fresh treatment or control and incubated at 37xc2x0 C. for 48 h. The slices were then washed in phosphate buffered saline and fixed in 2% glutaraldehyde (in 0.2M sodium cacodylate) for 5 min., following which they were washed in water and incubated in buffer for 5 min at 37xc2x0 C. The slices were then washed in cold water and incubated in cold acetate buffer/fast red garnet for 5 min at 4xc2x0 C. Excess buffer was aspirated, and the slices were air dried following a wash in water.
The TRAP positive osteoclasts were enumerated by bright-field microscopy and were then removed from the surface of the dentine by sonication. Pit volumes were determined using the Nikon/Lasertec ILM21W confocal microscope.